Selective NK3 Receptor Antagonists Research Articles

NK3 receptors mediate an increase in firing rate of midbrain dopamine neurons of the rat and the guinea pig

This in vitro study investigates and compares the effects of NK3 receptor ligands on the firing rate of rat and guinea pig midbrain dopamine neurons. The findings are discussed in the light of choosing suitable animal models for investigating pharmacological properties of NK3 receptor antagonists, which have been proposed to possess therapeutic activity in neuropsychiatric diseases like e.g. schizophrenia. In vitro midbrain slice preparations of both species were used to record (extracellularly) the firing rates of dopamine neurons located in the substantia nigra (SN) and ventral tegmental area (VTA). Furthermore, the effect of the D2 receptor agonist quinpirole on guinea pig SN and VTA dopamine neurons was investigated. The efficacy of quinpirole in inhibiting guinea pig dopamine neuron firing activity was much less as compared to that of rat dopamine neurons, suggesting a lower dopamine D2 autoreceptor density on the guinea pig neurons. The NK3 receptor agonist senktide induced in subpopulations of rat SN (55%) and VTA (79%) and guinea pig SN (50%) and VTA (21%) dopamine neurons an increase in firing rate. In responsive neurons this effect was concentration-dependent with EC₅₀ values of 3-5 nM (for both species). The selective NK3 receptor antagonist osanetant (100 nM) was able to partly block the senktide-induced increase in firing rates of dopamine neurons and shifted the concentration-response relation curves for senktide to the right (pA₂ values were ~7.5). The fractional block of the senktide responses by osanetant appeared to be larger in guinea pig dopamine neurons, indicating that osanetant is a more potent blocker of NK3 receptor-mediated responses with noncompetitive properties in the guinea pig.

Evidence for mediation of nociception by injection of the NK-3 receptor agonist, senktide, into the dorsal periaqueductal gray of rats

Ultrasound vocalizations (USVs) at approximately 22 kHz are usual components of the defensive response of rats. However, depending on the neural substrate that is activated, such as the dorsal periaqueductal gray (dPAG), USV emissions may be reduced. Activation of neurokinin-1 (NK-1)-mediated mechanisms of the dPAG causes analgesia, reduced 22 kHz USVs, and anxiogenic-like effects in rats exposed to the elevated plus maze (EPM). Involvement of other types of neurokinin receptors in this activation has not yet been evaluated. The present study examined whether local injections of the selective NK-3 agonist senktide (1-100 pmol/0.2 microL) into the dPAG can (1) cause anxiogenic effects in the EPM, (2) influence novelty-induced 22 kHz USVs, or (3) change nociceptive reactivity in the tail-flick test. Senktide elicited a significant increase in exploratory behavior, an effect accompanied by hyperalgesia and an increase in the number of 22 kHz USVs. The nociceptive effects, increased locomotor activity, and USV emissions elicited by local injections of senktide (50 pmol/0.2 microL) were reduced by prior injections of the selective NK-3 receptor antagonist SB222200 (50 pmol/0.2 microL) into the dPAG. These findings show that NK-3 receptors in the dPAG mediate nociceptive responses in this area, contrasting with the known fear-related processes mediated by NK-1 receptors in the dPAG. Both hyperalgesia and fear-related processes are accompanied by emissions of 22 kHz USVs.

Loperamide Inhibits Tachykinin NK3-Receptor-Triggered Serotonin Release Without Affecting NK2-Receptor-Triggered Serotonin Release From Guinea Pig Colonic Mucosa

The effect of loperamide on tachykinin NK2- and NK3-receptor-mediated 5-HT outflow from guinea pig colonic mucosa was investigated in vitro. The selective tachykinin NK2-receptor agonist [β-Ala8]-neurokinin A4–10 (βAla-NKA) or the selective NK3-receptor agonist senktide elicited an increase in 5-HT outflow from whole colonic strips, but not from mucosa-free muscle layer preparations. The enhancing effect of βAla-NKA and senktide was prevented by the selective NK2-receptor antagonist GR94800 or the selective NK3-receptor antagonist SB222200. Loperamide concentration-dependently suppressed the senktide-evoked 5-HT outflow, but failed to affect the βAla-NKA-evoked 5-HT outflow. The κ-opioid receptor antagonist nor-binaltorphimine or the δ-opioid receptor antagonist naltrindole displaced the concentration-response curve for the suppressant action of loperamide to the right without significant depression of the maximum. However, the µ-opioid receptor antagonist CTOP did not affect the suppressant effect of loperamide. We concluded that the NK3 receptor-triggered 5-HT release from colonic mucosa is suppressed by loperamide-sensitive mechanisms, whereas the NK2-receptor-triggered 5-HT release is loperamide-insensitive. Our data also suggest that the suppressant effect of loperamide is probably mediated by the activation of κ- and δ-opioid receptors located on intrinsic neurons.

Neurokinin NK1 and NK3 receptors as targets for drugs to treat gastrointestinal motility disorders and pain.

NK1 and NK3 receptors do not appear to play significant roles in normal GI functions, but both may be involved in defensive or pathological processes. NK1 receptor antagonists are antiemetic, operating via vagal sensory and motor systems, so there is a need to study their effects on other gastro-vagal functions thought to play roles in functional bowel disorders. Interactions between NK1 receptors and enteric nonadrenergic, noncholinergic motorneurones suggest a need to explore the role of this receptor in disrupted colonic motility. NK1 receptor antagonism does not exert consistent analgesic activity in humans, but similar studies have not been carried out against pain of GI origin, where NK1 receptors may have additional influences on mucosal inflammatory or "irritant" processes. NK3 receptors mediate certain disruptions of intestinal motility. The activity may be driven by tachykinins released from intrinsic primary afferent neurones (IPANs), which induce slow EPSP activity in connecting IPANs and hence, a degree of hypersensitivity within the enteric nervous system. The same process is also proposed to increase C-fibre sensitivity, either indirectly or directly. Thus, NK3 receptor antagonists inhibit intestinal nociception via a "peripheral" mechanism that may be intestine-specific. Studies with talnetant and other selective NK3 receptor antagonists are, therefore, revealing an exciting and novel pathway by which pathological changes in intestinal motility and nociception can be induced, suggesting a role for NK3 receptor antagonism in irritable bowel syndrome.

A tachykinin-like factor increases glutamate toxicity in rat cerebellar granule cells

Tachykinins (TKs), which include substance P, neurokinin A and neurokinin B, constitute a group of neuropeptides widely expressed in the CNS where they play several functions connected with neural modulation often in synergy with glutamate excitatory transmission. The aim of this study was to assess whether TKs modulate glutamate response of in vitro cultured cerebellar granule neurons and whether GSA (glutamate-sensitizing activity), a peptide released by these neurons, belongs to the TKs family. Treatment with substance P and other neurokinin 1 receptor (NK1) agonists does not affect the response of cerebellar granule neurons to glutamate toxicity. On the contrary, agonists neurokinin 2 receptor (NK2) and neurokinin 3 receptor (NK3) agonists increase, in a dose and time dependent fashion, the response of the same neurons to glutamate. MEN 10,627, a selective NK2 receptor antagonist, and (Trp 7,βAla 8) NKA (4–10), a selective NK3 receptor antagonist inhibit not only the sensitizing action to glutamate of their respective agonists. These antagonists almost equally reduce the glutamate-sensitizing activity of GSA. Such activity is also abolished in the presence of a polyclonal antibody directed against neurokinin B (NKB). These findings indicate that TKs increase glutamate sensitivity in cerebellar granule neurons and that the GSA previously detected in conditioned media of the same cultured neurons belongs to the TK family although its primary structure as compared to known TKs remains to be established.

Differential roles of spinal neurokinin 1/2 receptors in development of persistent spontaneous nociception and hyperalgesia induced by subcutaneous bee venom injection in the conscious rat

To evaluate the roles of spinal neurokinin receptors in the development of persistent nociception and hyperalgesia to thermal and mechanical stimuli induced by subcutaneous (s.c.) bee venom injection, effects of intrathecal (i.t.) pre- or post-treatment with a non-selective antagonist of (NK1/2) receptors, [D-Arg1,D-Trp7,9,Leu11] substance P (spantide), and a selective NK3 receptor antagonist, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methyl acetamide (SR142801) were assessed in conscious rat. Injection of bee venom s.c. into the plantar surface of one hind paw resulted in a pathological pain phenomenon characterized by a 1–2h single phase of persistent spontaneous nociceptive behaviors (continuously flinching the injected paw) and a 72–96h profound primary thermal and mechanical hyperalgesia in the injection site and a secondary thermal hyperalgesia in the non-injected hindpaw. Pre-treatment with spantide i.t. at 0.05μg, 0.5μg and 5μg produced a dose-related suppression of the bee venom-induced flinching reflex during the whole time course and the inhibitory rate was 24±12.60% (35.38±4.12 flinches/5min, n=5), 48±6.75% (24.53±2.90 flinches/5min, n=5) and 60±7.69% (18.88±3.58 flinches/5min,n =5) respectively when compared with the saline control group (46.80±2.60 flinches/5min,n =5). Post-treatment of spantide i.t. at the highest dose (5μg) used in the present study 5min after bee venom injection also produced a 49% suppression of the flinching reflex in the control group [post-spantide vs saline: 19.42±3.15 (n=5) vs 38.42±3.25 flinches/5min (n=5)]. Moreover, i.t. pre-treatment with 5μg spantide partially prevented the primary and secondary thermal hyperalgesia from occurring, while it did not show any influence on the development of primary mechanical hyperalgesia. Neither the established thermal nor mechanical hyperalgesia identified in the above sites was affected by i.t. post-treatment with the same dose of spantide 3h after bee venom injection. Pre and post-treatment of SR142801 did not produce any significant effect on the bee venom-induced spontaneous pain and thermal and mechanical hyperalgesia. Our present result suggests that activation of spinal NK1/2 receptors is involved in both induction and maintenance of the persistent spontaneous nociception, while it is only involved in induction of the primary and secondary thermal, but not primary mechanical hyperalgesia induced by s.c. bee venom injection. The spinal NK3 receptor seems not likely to be involved in the bee venom-induced behavioral response characterized by spontaneous pain and thermal and mechanical hyperalgesia.

Pharmacologic characterization of receptor types mediating coronary vasodilator actions of sensory neuropeptides in the guinea pig.

The receptor types mediating sensory neuropeptide-induced coronary vasodilatation were elucidated on isolated guinea pig hearts perfused with isotonic buffer containing 20 mM KCl. Substance P and the selective neurokinin-1 (NK1) receptor agonist [Sar9, Met(O2)11]-substance P produced dose-dependent reductions in perfusion pressure, but the selective NK2 receptor agonist [Nle10]-neurokinin A4-10 and the selective NK3 receptor agonist [MePhe7]-neurokinin B produced no change. The vasorelaxant effects of substance P and the NK1 receptor agonist were abolished by the selective NK1 receptor antagonist FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N-methy l-N-phenylmethyl-3-(2-naphthyl)-L-alaninamide), whereas the selective NK2 receptor antagonist SR48968 ((S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl )-butyl] benzamide) and the selective NK3 receptor antagonist SR142801 ((S)-(N)-( 1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)4-p henylpiperidin-4-yl)-N-methylacetamide) produced partial inhibition on their responses. Calcitonin gene-related peptide (CGRP) produced dose-dependent vasodilatation on the guinea pig coronary blood vessels, which was significantly (p = 0.0067) inhibited by the selective CGRP1 receptor antagonist hCGRP8-37. The selective CGRP2 receptor agonist [Cys(acetomethoxy)2,7]CGRP had no effect on perfusion pressure. These results demonstrate that the sensory neuropeptides substance P and CGRP are effective vasodilators of the guinea pig coronary vascular bed. The receptor types mediating their vasorelaxant effects were identified to be the NK1 receptors and CGRP1 receptors, respectively.

Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum.

1. The role of NK1 and NK3 receptors in synaptic transmission between myenteric neurons during motility reflexes in the guinea-pig ileum was investigated by recording intracellularly the reflex responses of the circular muscle to distension or compression of the mucosal villi. Experiments were performed in a three-chambered organ bath that enabled drugs to be selectively applied to different sites along the reflex pathways. 2. When applied in the recording chamber, an NK1 receptor antagonist, SR140333 (100 nM), reduced by 40-50% the amplitudes of inhibitory junction potentials (i.j.ps) evoked in the circular muscle by activation of descending reflex pathways. This effect was abolished when synaptic transmission in the stimulus region was blocked with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+, leaving only the component of the descending reflex pathway conducted via long anally directed collaterals of intrinsic sensory neurons. 3. SR140333 (100 nM) had no effect on descending reflex i.j.ps when applied to the stimulus region. Ascending reflexes were also unaffected by SR140333 in the stimulus region or between the stimulus and recording sites. 4. Septide (10 nM), an NK1 receptor agonist, enhanced descending reflexes by 30-60% when in the recording chamber. [Sar9,Met(O2)11]substance P had no effect at 10 nM, but potentiated distension-evoked reflexes at 100 nM. 5. A selective NK3 receptor antagonist, SR142801 (100 nM), when applied to the stimulus region, reduced the amplitude of descending reflex responses to compression by 40%, but had no effect on responses to distension. SR142801 (100 nM) had no effect when applied to other regions of the descending reflex pathways. 6. SR142801 (100 nM) only inhibited ascending reflexes when applied at the recording site. However, after nicotinic transmission in the stimulus region was blocked, SR142801 (100 nM) at this site reduced responses to compression. 7. Contractions of the circular muscle of isolated rings of ileum evoked by low concentrations of septide, but not [Sar9,Met(O2)11]substance P, were potentiated by tetrodotoxin (300 nM). 8. Contractile responses evoked by an NK3 receptor agonist, senktide, were non-competitively inhibited by SR142801. After excitatory neuromuscular transmission was blocked, senktide produced inhibitory responses that were also antagonised by SR142801, but to a lesser extent and in an apparently competitive manner. 9. These results indicate that tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons. NK3 receptors play a role in transmission from intrinsic sensory neurons and from ascending interneurons to excitatory motor neurons during motility reflexes.

Further Evidence that Central Tachykinin NK-1 Receptors Mediate the Inhibitory Effect of Tachykinins on Angiotensin-Induced Drinking in Rats

Polidori, C., R. Ciccocioppo, G. De Caro and M. Massi. Further evidence that central tachykinin NK-1 receptors mediate the inhibitory effect of tachykinins on angiotensin-induced drinking in rats. Peptides 19(1) 149–155, 1998.—The order of potency of tachykinin (TK) receptor agonists suggests that TK NK-1 receptors mediate their inhibitory effect on water intake induced by intracerebroventricular (ICV) injection of angiotensin II (AngII) in rats. The present study was aimed at further evaluating which TK receptor subtype mediates the effect, using selective antagonists for the TK receptor subtypes. Pulse ICV injection of the TK agonist neuropeptide γ (NPγ), 31–250 ng/rat, markedly inhibited AngII-induced water intake. The ICV injection of the NK-1 receptor antagonist SR140333, 0.5 μg/rat, significantly reduced, while 1 μg/rat completely abolished the inhibitory effect of NPγ, 125 ng/rat. The selective NK-2 receptor antagonist SR48968 and the selective NK-3 receptor antagonist R820 were devoid of any effect up to the ICV dose of 2 μg/rat. On the other hand, ICV injection of SR140333, 1 μg/rat, did not increase drinking induced by ICV injection of AngII, 0.1–10 ng/rat, and did not increase drinking in water sated or water deprived rats. The results of the present study confirm that central TKergic mechanisms inhibit AngII-induced drinking in rats, and provide further evidence that TK NK-1 receptors mediate the effect. Failure of ICV injected SR 140333 to increase AngII-induced drinking, as well as water intake in sated or deprived rats suggests that brain NK-1 receptor mechanisms apparently do not exert a tonic control on AngII-induced drinking and, in general, on water intake in rats. From a pharmacological point of view, the inhibitory effect of TKs on the dipsogenic action of AngII can represent a functional test for activity at central NK-1 receptors in rats.

Transglutaminase-synthesized spermine derivative of substance P recognizes rat portal vein neurokinin-3 receptors.

The effects of the transglutaminase-synthesized polyamine derivatives of Substance P (SP) have been further characterized by their ability to contract in vitro the rat portal vein strip (RPV), a pharmacological preparation particularly rich in NK-3 receptors. The effects of selective agonists of NK-1, NK-2 and NK-3 receptors [Sar9,Met(O(2))11]SP, beta-Ala8 NKA(4-10), and senktide respectively, were also evaluated by measuring RPV concentration-response curves. Peptide [GR-82334 (NK-1) and MEN-10,376 (NK-2)] and nonpeptide [WIN 51,708 (NK-1) and SR 142801 (NK-3)] NK receptor antagonists were used to confirm the participation of the different NK receptors to contractile response. Our results demonstrated that the spermine derivative of SP (Spm-SP), previously shown to be unable to recognize NK-1 and NK-2 receptors in some bioassays, contracts RPV (EC50 = 588 nM) better than the native neuropeptide (EC50 = 1120 nM). A pretreatment with thiorphan, an inhibitor of neutral endopeptidases, significantly reduced such a difference. While this inhibitor shifts the SP concentration-response curves to the left (EC50 = 720 nM) the action of Spm-SP and [Sar9,Met(O(2))11]SP were completely thiorphan-resistant. In the absence of thiorphan we found the following rank order of potency: senktide > > beta-Ala8 NKA(4-10) > [Sar9,Met(O(2))11]SP = Spm-SP > SP. Among the mentioned NK receptor antagonists, only the selective NK-3 receptor antagonist, SR 142801, shifted to the right Spm-SP and [Sar9,Met(O(2))11]SP concentration-response curve, showing pKB values of 5.84 and 5.88, respectively. Therefore, the reported results suggest that the introduction of a Spm moiety into the SP alters the parent peptide molecule by increasing its affinity for NK-3 receptors and/or by preventing its degradation by some proteolytic enzymes.

Characterization of NK3 receptors in rabbit isolated iris sphincter muscle.

1. Tachykinin NK3 receptors were characterized in the rabbit isolated iris sphincter muscle by use of autoradiography and in vitro functional studies. 2. [125I]-[MePhe7]-neurokinin B (NKB) (1nM), a selective NK3 receptor agonist, specifically labelled a population of NK3 receptors that were uniformly distributed throughout the rabbit iris sphincter muscle. This labelling was inhibited by unlabelled [MePhe7]-NKB (1 microM) but not by the NK1 receptor antagonist CP 99994 (1 microM). 3. In the presence of CP 99994 (1 microM), the selective NK3 receptor agonists senktide (n = 14) and [Pro7]-NKB (n = 4), and the natural preferred ligand for the NK3 receptor, NKB (n = 8), were potent contractile agents in the rabbit iris sphincter muscle. They all produced monophasic concentration-effect curves with pD2 values of 9.53 +/- 0.08, 8.56 +/- 0.09 and 9.75 +/- 0.09, and nH values of 0.93 +/- 0.03, 1.53 +/- 0.17 and 0.76 +/- 0.06, respectively. [MePhe7]-NKB (n = 12) was also a potent agonist, but produced shallow concentration-effect curves which appeared biphasic (nH = 0.45 +/- 0.04). 4. Contractile responses to senktide were surmountably antagonized in a concentration-dependent manner by the selective non-peptide NK3 receptor antagonist, SR 142801 (3-30 nM; pA2 = 8.9; slope = 0.99) and the non-peptide NK2/NK3 receptor antagonist, SR 48968 (3-30 microM; pA2 = 6.1; slope = 1.5). These pA2 values were consistent with functional rabbit NK3 receptors more closely resembling guinea-pig and human NK3 receptors, than rat NK3 receptors. SR 142801 (10-100 nM) and SR 48968 (3 and 30 microM) inhibited responses to low (< or = 1 nM) but not higher (> 1 nM) concentrations of [MePhe7]-NKB, and concentration-effect curves to [MePhe7]-NKb became steeper and monophasic in the presence of either antagonist. 5. SR 142801 (3-30 nM) and SR 48968 (3-30 microM) also surmountably antagonized concentration-effect curves to [Pro7]-NKB and NKB, although results were more difficult to interpret, since the relationship between log concentration-ratios and the concentration of antagonist used did not adhere to the Schild equation. However, analysis of data with the lowest concentration of SR 142801 (3 nM) tested against NKB, and SR 48968 (3 microM) tested against [Pro7]-NKB and NKB, yielded apparent pA2 estimates of 9.3, 6.8 and 6.4, respectively, consistent with blockade of NK3 receptors. 6. SR 142801 (100 nM) had no effect on contractions induced by transmural nerve stimulation (2 Hz, 0.3 ms, 20 V for 30 s), whereas CP 99994 (1 microM) abolished these responses. 7. Phenoxybenzamine pretreatment (20 microM, 10 min) markedly reduced maximum responses to [MePhe7]-NKB (from 101 +/- 6.2% to 38 +/- 9.5% reference contraction, n = 4) and induced a marked (10 fold) rightward shift in the concentration-effect curve. The residual responses to [MePhe7]-NKB after phenoxybenzamine pretreatment were unaffected by 1 microM CP 99994 (maximum response = 41 +/- 9.4%, n = 4). 8. These results demonstrate autoradiographically and functionally, the presence of NK3 receptors in rabbit iris sphincter muscle that mediate contractile responses to NK3 receptor agonists, but not to sensory trigeminal nerve simulation. The present data with senktide and selective NK3 receptor antagonists suggest that functional rabbit NK3 receptors more closely resemble human and guinea-pig NK3 receptors than rat NK3 receptors. However, the pharmacological profiles of [MePhe7]-NKB, SR 142801 and SR 48968 suggest the presence of an 'atypical' NK3 receptor or a heterogeneous population of NK3 receptors in this tissue.

Investigation into the 5-hydroxytryptamine-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon.

1. The aim of this study was to characterize the receptors mediating the atropine-resistant neurogenic contraction to 5-hydroxytryptamine (5-HT) in the longitudinal muscle of the guinea-pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5-HT by the use of selective antagonists. 2. In the presence of atropine (0.3 microM), guanethidine (5 microM), hexamethonium (100 microM), ketanserin (0.1 microM) and indomethacin (3 microM), 5-HT (0.01-3 microM) produced concentration-dependent neurogenic contractions of colonic strips and at 0.3 microM produced a maximal effect (pEC50 = 7.39 +/- 0.09, n = 18). The 5-HT4 receptor stimulant, 5-methoxytryptamine (5-MeOT, 0.03-10 microM) also produced neurogenic contractions with similar maximum effect to those of 5-HT (pEC50 = 6.89 +/- 0.16). 3. The 5-HT4 receptor antagonist, DAU 6285 (3 microM) shifted the concentration-response curves to both 5-HT and 5-MeOT to the right without significant depression of the maximum, but the 5-HT1/5-HT2 receptor antagonist, metitepine (0.1 microM) and the 5-HT3 receptor antagonist, ondansetron (0.3 microM) had no effect on the control curves to 5-HT and 5-MeOT. 4. The selective NK1 receptor antagonist, FK 888 (1 microM) markedly attenuated the contractions to 5-HT and 5-MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5-HT and 5-MeOT. 5. These results indicate that the 5-HT-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon is due to activation of 5-HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5-HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5-HT4 receptor-mediated contraction is evoked indirectly via tachykinin release from tachykinin-releasing excitatory neurones.